In an effort to facilitate the use of known denaturant stable proteases by investigators involved in the structural characterization of proteolysis-resistant peptides and proteins, the stability and cleavage specificity in denaturant of five proteases reported to be denaturant stable will be determined. Following purification of these denaturant stable enzymes, appropriate chromophoric peptide substrates (p-nitroanilides) will be cleaved by the denaturant stable protease In the presence and absence of denaturant. The Km, Vmax, Vmax/Km and kcat values of each of these enzymes for its corresponding substrate will be determined in the presence and absence of denaturant. The integrity of the known specificity of each protease will be assessed in the presence of denaturant by proteolysis on two types of protein substrates. First, small peptides of known amino acid sequence will be cleaved by a denaturant stable protease In the presence and absence of denaturant. The peptide fragments will be separated by reverse phase high performance liquid chromatography and quantitatively identified by amino acid compositional analysis and N-terminal amino acid analysis by dansylation. Next, the cleavage specificity of these enzymes in the presence of denaturant will be investigated using larger proteins as substrates. These larger proteins present a wider variety of peptidyl bonds with which to detect departures from known cleavage specificities. The cleavage sites will be quantitatively identified as indicated above. Immobilization of the stable proteases will be used in an attempt to increase the denaturant stability and decrease autolysis. Knowledge of the predictability of cleavage preference for these unusual proteases will greatly enhance their utility in the site-specific fragmentation of insoluble or proteolysis-resistant proteins and polypeptides.